Biology

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Trait selection and the adaptive potential of 𝘗𝘪𝘤𝘦𝘢 𝘮𝘢𝘳𝘪𝘢𝘯𝘢 in the face of climate change
(University of Waterloo, 2024-12-04) Henry, Sabina
The local persistence of long-lived organisms is at risk as climate change drives a rapid shift in selection regimes world-wide. Although adaptive evolution is one of the main mechanisms by which populations persist in changing environments, we have little information regarding how selection regimes will shift in response to continued climate change, nor on the potential for trees to evolve adaptively under novel selection pressures. To address these gaps, here we assessed the changes in selection in three sites along a spatial climate gradient which mimics expected temporal changes in climate and determined whether trait covariance might accelerate or impede the rate of adaptive evolution of seven P. mariana populations in the warmer and drier environment. In three common garden sites established 50 years ago, we measured an array of traits which represent water use, response to temperature, structural investment, and metabolic efficiency. Our findings reveal that all 10 traits measured in this study were under selection in at least one site. We also find different traits are under selection in each site, with four instances where the shift in selection gradient is consistent with shifts in climate: water use efficiency (WUE); needle carbon to nitrogen ratio (CN); the interaction between WUE and CN; and the interaction between CN and huber value. In the warm and dry site, traits under selection were largely uncorrelated, with only four of the 49 trait combinations under selection exhibiting intra population trait covariances. The shifts in selection gradient suggest that climate change may select for needles with higher WUE, higher structural carbon and higher hydraulic supply to the needles. The few trait-trait correlations indicate that phenotypic integration should neither impede nor facilitate adaptive evolution, leaving P. mariana provenances with the evolutionary flexibility to respond to climate change regardless of the direction to selection.
RNA-seq exploration of host responses and viral genomic diversity in SARS-CoV-2 infection
(University of Waterloo, 2024-11-18) Luc, Jessica
Since the discovery of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there have been over 700 million cases and over 7 million deaths worldwide. Although some patients develop severe symptoms such as multiple system organ failure, others can be asymptomatic. This reflects the role of the immune system in disease progression. Although there exist standard PCR and antigen-based tests for SARS-CoV-2 detection and diagnostics, an important research goal is the identification of predictive biomarkers that are indicative of COVID-19 severity. In this thesis, I performed bioinformatic analyses of two original RNA-seq datasets generated by collaborators to gain insights into the mechanisms and host responses of SARS-CoV-2 infection. The first dataset consisted of transcriptomic data from SARS-CoV-2 infected human lung cells as well as bat (Eptesicus fuscus) derived cells, providing insights into host responses of multiple mammalian species. The second dataset consisted of transcriptomic data from nasopharyngeal swabs collected from COVID-19 patients. A bioinformatic approach was used in both datasets to identify host genes that were up-regulated and down-regulated by SARS-CoV-2 infection as well as to identify biologically relevant viral mutations. The analysis resulted in the discovery of a novel R685P mutation in the SARS-CoV-2 spike glycoprotein, that had increased frequency in the Eptesicus fuscus derived cell line. Variant analysis from the clinical nasopharyngeal swab RNA-seq dataset also revealed the existence of a SARS-CoV-2 quasispecies containing multiple distinct viral genotypes with a unique population structure in each patient. Despite each dataset being distinct, a similar bioinformatic analysis was applied to both datasets to reveal host response patterns and visualize the evolution and mutational spectrum of SARS-CoV-2. Although COVID-19 is no longer considered a global emergency, furthering our knowledge of the SARS-CoV-2 infection can aid in the development in new therapeutics and diagnostic tools.
Metal Concentrations in an Arctic Wastewater Wetland: Insights and Innovations from Baker Lake, Nunavut, Canada
(University of Waterloo, 2024-11-14) McPhedran, Bronte
The unique climatic conditions and isolated geographies of Arctic communities present distinct challenges for municipal wastewater treatment. Most communities depend on natural wetlands to provide passive treatment of wastewater; however, increased pressure on these systems from prolonged use, climate change, and intensifying human activity has resulted in the recent development of northern-specific standards for wastewater. These standards, among other objectives, aim to maintain healthy fish populations and diverse aquatic ecosystems, while calling for community consultation, integration of traditional knowledge, and adherence to federal and territorial regulations. A passive wastewater treatment system near Baker Lake, NU, served as a case study to address knowledge gaps regarding ecosystem function and trace metal dynamics in abiotic and biotic ecosystem compartments subject to wastewater inputs. Additionally, this research explored a method for time-resolved analysis of contaminant histories, which is particularly valuable in environments lacking baseline data or where regular monitoring is challenging, including in the Arctic. Between the summers of 2019 and 2021, a series of lakes that represented wastewater-affected and reference systems were sampled for water, sediment, and fish. Water from wastewater-affected lakes had elevated concentrations of metals (e.g., Mn, Fe, Cu) and increased primary productivity (as inferred from chlorophyll-a concentrations), with persistent hypereutrophic conditions; extremely low dissolved oxygen levels were recorded in the lake closest to wastewater influent. Although sediment analysis near wastewater sources indicated higher levels of Cu and Zn, overall sediment chemistry was similar across all sites. The influence of wastewater on the trophic ecology of Burbot (Lota lota) was minimal, whereas Arctic grayling (Thymallus arcticus) had more enriched (i.e., less negative) δ13C at wastewater-affected sites, suggesting altered energy pathways in lakes that receive nutrients from wastewater. Analyses of metals in muscle tissue revealed lower, although not significant, concentrations of methylmercury – the toxic, biomagnifying form of mercury - in fish from wastewater-affected sites compared to those from reference sites. These lower concentrations of methylmercury in fish muscle tissue occurred despite higher concentrations in abiotic media, potentially because of somatic growth dilution, but more research is necessary. Analyses of trace metals in otoliths revealed distinct multi-element otolith signatures (i.e., Zn, Mn, Cu, Fe, and Pb) between wastewater-affected and reference lakes, and significantly greater concentrations of Fe in a lake receiving wastewater across two reconstructed periods of growth. Together, findings from this research not only fill knowledge gaps regarding the influence of municipal wastewater on cold, oligotrophic freshwater systems in the subarctic, but also highlight the potential uses and limitations of otoliths as markers of wastewater exposures.
The evolutionary and ecological factors that shape ectoparasite populations and communities at multiple scales
(University of Waterloo, 2024-11-12) Sauk, Alexandra
Although parasites are one of the most prolific and diverse consumer groups on the planet, they are often excluded from biodiversity surveys as they are difficult to detect and identify. This deficit limits our understanding of host-parasite relationships and parasite diversity. The vast diversity of host-parasite relationships means that many ecological and evolutionary forces may be at play, shaping the evolution of host and parasite in different ways and varying between species pairs. Bats and their ectoparasites provide a unique system to study the factors influencing parasite populations and communities. The different ecological niches and social behaviours of bats provide variation in the ectoparasites they encounter, and the selective forces experienced by the ectoparasites. I hypothesised that certain attributes of ectoparasite infections (e.g., ectoparasite diversity and infection level) are influenced by the life history traits of both host and ectoparasite and be the environmental restrictions of individual ectoparasite species. Using a collection of ectoparasites passively collected from bats throughout Atlantic Canada between 1999 and 2017, I quantified the ectoparasite communities of two bat species, Myotis lucifugus and M. septentrionalis, and used model-based inferences to assess the differences in infection of their two most common ectoparasites, the mite Spinturnix americanus and the flea Myodopsylla insignis. I found that both bat species had similar ectoparasite communities while S. americanus and M. insignis showed opposing trends in presence and abundance between the two bat species, in keeping with their different life history strategies. I also used a subset of this collection to compare how life history traits and host-parasite dynamics influence the genetic structure and biogeography of co-infecting ectoparasites. I found limited genetic structure with M. insignis exhibiting some isolation by distance between Labrador and Nova Scotia and S. americanus exhibiting regional differentiation between the island of Newfoundland and the mainland. I also provide a synthesis of the currently described bat ectoparasites in North America and an analysis of how host characteristics and environmental factors influence ectoparasite richness and geographic distribution. I found that estimated ectoparasite richness varies widely between host species but is influenced by sampling effort. Bat ectoparasite diversity appears consistent with the predictions of the latitudinal diversity hypothesis with a 3.4% decrease in species richness for every degree increase in latitude. Overall, my findings add to the evidence that ectoparasite populations and communities are shaped by life history traits of the host and ectoparasite. I suggest multidisciplinary collaborations between bat biologists, parasitologists, and taxonomists are necessary to collect ectoparasites and catalogue bat-ectoparasite associations to better understand the ecological and evolutionary forces that shape these communities and to better be able to conserve them in the face of ongoing threats from climate change and landscape changes.
Predicting Black Carp (Mylopharyngodon piceus) age at sexual maturity with water temperature
(University of Waterloo, 2024-09-24) Wu, Jiayi
Black Carp is one of the four invasive Asian carps in North America. It is currently considered established in the Mississippi River and has the potential to invade into the Great Lakes watershed. Black Carp is known for its ability to consume freshwater unionids, and thus could bring potential changes to the benthic ecosystems. For effective management and mitigation strategies, it is critical to understand its likely population growth rate in new environments, and to be able to predict the environmental conditions of the potential habitats it may become established in. Age at sexual maturity of the population has been identified as an important variable in determining the population establishment speed, as faster maturation typically corresponds to faster population growth. I demonstrated that temperature metrics such as air temperature can be used to predict the age of maturity of Black Carp. Using age at maturity data and related climate metrics around the world, I found that winter duration and winter air temperature are the two best predictors of Black Carp age at maturity. This suggests that winter can play an important role in the maturation process of this invasive species. Populations experiencing longer and colder winters tend to mature later. While temperature may be an important predictor for the speed of Black Carp invasion, water temperature data is not always available for regions of interest. Using empirical water temperature from 12 tributary locations in the Great Lakes watershed, I established a simple statistical model to predict water temperature during the growing season. I found that simple regression-based models using air temperature as input can perform better than a complex processed-based global model for estimating water temperature in the Great Lakes region.
Effect of Riparian Forest Cover on Cellulose Decomposition in Agricultural Streams
(University of Waterloo, 2024-09-24) Hewitt, Kristen
Agricultural practices often result in human impacts to aquatic ecosystems. The presence of riparian forests has been shown to mitigate some of these impacts, however, there is limited knowledge of how riparian forests can mitigate human impacts on microbial community function. This study investigates effects of riparian forest cover on in-stream cellulose decomposition. The study used the cotton strip assay to compare two sets of streams in summer, fall, and winter seasons to assess the association between riparian forest cover and cellulose decomposition in summer in agricultural catchments. Results from summer and autumn show there is greater cellulose decomposition in streams with riparian forest cover than streams without riparian forest cover. Cellulose decomposition also increased linearly with riparian forest in the summer. Decomposition in the assessed streams was also compared to cellulose decomposition in reference streams within the same region. Significant differences were observed between reference and non-forested streams, but not reference and forested streams, in the summer. These differences were not explained by typical environmental parameters (e.g., temperature, nutrients). These results suggest that riparian forest cover influences the decomposition by protecting organic matter processing in streams, though the specific threats against which riparian forests provide protection remain unclear. In future studies, considering agricultural contaminants (e.g., pesticides, pharmaceuticals) and investigating the differences in microbial community composition between groups may help close this knowledge gap.
Ploidy impacts the response of Chinook salmon (𝘖𝘯𝘤𝘰𝘳𝘩𝘺𝘯𝘤𝘩𝘶𝘴 𝘵𝘴𝘩𝘢𝘸𝘺𝘵𝘴𝘤𝘩𝘢) to pathogen and thermal stress
(University of Waterloo, 2024-09-23) Cadonic, Ivan
As climate change continues to reduce wild salmon populations, global reliance on aquaculture will become increasingly essential to sustain food availability. However, the environmental implications of aquaculture in causing Pacific salmon stock declines are becoming more apparent and as such the industry needs to transition to practices that would reduce negative impacts on wild fishes. Widespread utilization of sterile triploid fishes offers an alternative to normal diploid fish since they are unable to breed with wild population and are less likely to interact with wild fishes. Triploid salmonids have an extra set of chromosomes that prevent them from producing viable gametes, while also maintaining flesh quality for longer due to lack of nutrient deposition from muscle to eggs during sexual maturation. When farmed under optimal conditions, triploid fish perform similarly to diploids; however, when conditions become suboptimal (e.g. high temperature & infection with pathogens) triploids have reduced survival. The reasons for this reduced performance are mostly unknown, so this thesis aimed to investigate the molecular impacts of increased ploidy in Chinook salmon (Oncorhynchus tshawytscha). Specifically, the response of microRNA (miRNA) and protein coding mRNAs were investigated before and after exposure to pathogenic and thermal stress. MiRNAs are small, non-coding RNAs that bind to specific mRNA molecules and reduce protein translation. I hypothesized that diploid and triploid salmonids have subtle differences in molecular (miRNA, mRNA) indices that are exacerbated by changes in the environment (pathogens and temperature), which results in their poorer performance in response to stress. Chapter 2 investigated the miRNA responses in three immune tissues (hindgut, head kidney, and spleen) before and after exposure to Vibrio anguillarum, a common pathogen Chinook experience in open-sea net pens. Overall, ploidy had minimal impacts on miRNA abundances prior to stress. Abundance of miRNAs were altered in both ploidies after Vibrio exposure to support immune function. Just prior to Vibrio-induced mortality, the spleen of triploid fishes had altered miRNA abundances which identified that in triploids, the spleen is likely associated with their reduced immune competence. Chapter 3 investigated the immune mRNA responses in gill and heart ventricle before and after recovery from acute thermal stress. Triploid gills likely have altered pathogen sensing based on reduced pattern recognition receptor mRNA abundance. Tissue specific alterations after thermal stress provided insight into immune dynamics during a cellular heat-shock response. Specifically, pro-inflammatory cytokine mRNA abundance was elevated after recovery from thermal stress. The immune genes measured are likely thermally responsive in a time-dependent manner based on their variability after recovery from thermal stress. Chapter 4 investigated the cardiorespiratory impacts of increased ploidy before and after a critical thermal maximum (CTmax) trial. In agreement with previous studies, triploid hearts became arrhythmic at lower temperatures indicating that their hearts are less resilient to acute changes in temperature. Additionally, ventricle expression of cardiorespiratory genes was altered in triploid fish, implicating that this tissue is associated with their reduced thermal performance. Overall, this thesis provides evidence for the molecular dysregulation associated with increased ploidy in Chinook salmon. These impacts are minimal under normal conditions but exacerbated by pathogenic and thermal stress. The results here provide targets for aquaculture to develop breeding programs or therapeutics that can be used to improve health and well-being of salmonids, including triploids, in response to climate change.
Genome-encoded metabolic potential of the Nitrosocosmicus genus and related ammonia-oxidizing archaea
(University of Waterloo, 2024-09-17) Cornell, Calvin
Since the discovery of ammonia-oxidizing archaea (AOA), genomic and experimental evidence suggests that mixotrophic growth may complement chemolithotrophic ammonia oxidation for some AOA representatives. Although members of the Candidatus Nitrosocosmicus genus have been implicated in the use of alternate sources of energy and carbon, stronger genomic evidence is needed to support testing of hypotheses with robust experimental design. This study involved analysis of 58 genomes spanning all four known AOA groups (i.e., I.1a, I.1b, 1.1a-associated, thermophilic AOA; ThAOA). The analyses focused primarily on transporters and enzymes, linking the former to potential substrates from the transporter classification database (TCDB) and the latter to KEGG, Carbohydrate-Active enZYmes (CAZymes), and pathways from GapSeq. Correlated with genome size, the results demonstrate that genomes of the I.1b group, including those from the Ca. Nitrosocosmicus genus, showed the highest abundances of exclusive (only found within this group) and preferred (primarily associated with this group) proteins for alternative metabolism, followed by ThAOA, I.1a, and I.1a-associated. There was extensive functional heterogeneity among representative genomes, particularly for Ca. Nitrosocosmicus and Nitrososphaera genera. The group I.1b genomes encoded for the potential to use nitrogenous substrates such as urea, alanine, glycine, asparagine, and glutamine. Although Ca. Nitrosocosmicus genomes were more commonly associated with use of TCA cycle intermediates, such as citrate, malate, oxaloacetate, and succinate, this potential was likewise found in ThAOA and I.1a representatives. Several I.1b representative genomes encoded enzymes for metabolism of ureidoacrylates and carbamate, CAZymes associated with glycoside hydrolases, genes implying oxidation of sulfur and manganese, and polyamine metabolism. However, genes for synthesis and degradation of polyamines were common to many AOA genomes analyzed. Overall, the results suggest that I.1b-associated representatives encode for mixotrophic metabolism, and future research should verify associated substrate predictions experimentally. Given broad distributions of AOA within terrestrial and aquatic environments, these findings have implications for biogeochemical cycling of carbon and nitrogen on a global scale.
The Effect of Corn Starch and Kappa-Carrageenan Probiotic Encapsulation on Growth and Immune Response in Chinook Salmon (Oncorhynchus tshawytscha)
(University of Waterloo, 2024-09-16) Latimer, Maureen
As vaccines have limited efficacy in fish, alternative preventative measures are used to combat infectious diseases in aquaculture, including probiotics. To be effective, a probiotic must remain stable during storage, feeding, and transit through the gastrointestinal tract. Thus, preparing probiotic feed requires the use of a coating agent and several different types have been tested, as some have adverse effects. To better understand the effect of corn starch and kappa () -carrageenan as a combined coating agent on growth and immune performance in Chinook salmon, a growth trial was conducted. Three different treatments were used, including regular feed, regular feed with a corn starch and -carrageenan coating, and probiotic feed with the same coating. The probiotic feed included a mixture of BioPower® PA (Pediococcus acidilactici, strain CNCM MA 18/5M; Lallemand Inc., Canada) and Seed 14 (Limosilactobacillus reuteri LRE2; Probiotical SpA, Italy). Sampling timepoints were at 0-, 1-, 3-, 7-, and 28-days post diet-introduction, and at each sampling timepoint, the weight and length of 6 fish per treatment were measured, and the head kidney, spleen, and hindgut were collected. There was an additional timepoint at Day 42 where the weight and length of the remaining fish in each barrel were recorded, but no tissues were collected. To assess immunological effects of the coating agent, qPCR for IL-1, IL-8, and TGF- was performed on spleen, head kidney, and hindgut samples. In the hindgut, additional genes were assessed to determine changes in gut barrier integrity, including E-cadherin, claudin 15, junctional adhesion molecule 1 alpha, mucin 2, tricellulin, villin 1, and zonula occludens-1. No significant differences in weight or length were observed between treatments measured over 42 days. No significant differences were found in the assessed proinflammatory transcript levels in the spleen or head kidney sampled at 0-, 1-, 3-, 7-, and 28-days post diet-introduction. Transient changes in the regulatory cytokine TGF- were seen in these tissues. In the hindgut, upregulation of all three immune transcripts were seen on Day 7 in both coated treatments, but these changes were transient, with no significant differences seen on Day 28. Transient changes were also seen in gut barrier gene expression, with no significant differences seen between treatments on Day 28. These results suggest that corn starch and -carrageenan as a combined coating agent has no short-term negative effects on the growth or immune status of Chinook salmon and that it can be used to deliver probiotics that will improve the health of fish in aquaculture.
Effects of Trastuzumab on Alternative Splicing in HER2+ BT474 Breast Cancer Cells
(University of Waterloo, 2024-09-12) Piticaru, Benjamin
Alternative splicing (AS) is a fundamental process that enhances transcriptomic diversity and protein isoform complexity in eukaryotic cells. Deregulation of AS plays a critical role in various diseases, particularly cancer, where erroneous splicing patterns contribute to oncogenesis, tumor progression, and therapeutic resistance. Human epidermal growth factor receptor (HER)2, overexpressed in more than 20% of breast cancers, has clinical relevance in AS. The humanized monoclonal antibody (mAb) Trastuzumab has been used for decades to fight HER2+ breast cancer with positive results. However, there are many unknowns surrounding the mechanism of this drug and the effects it has on alternative splicing. Next generation long read sequencing technologies like Oxford Nanopore allow researchers to sequence full length transcripts. The ability to sequence long intron/exon spans and repeating regions enables long read sequencing technology to provide new insights into the AS patterns of genes. Identifying AS events is important for understanding changes in function and pathways affected by differing conditions (e.g., drug treatment, diseased cells). Software tools tailored to long read data like Long-read Isoform Quantification and Analysis (LIQA), Full-Length Alternative Isoform Analysis of RNA (FLAIR), FLAME, and Nanosplicer enhance data analysis abilities to detect AS events. They analyze sequencing data by mapping reads to genomes, identifying splice junctions, and clustering AS events. These tools then compare AS events between conditions (e.g., treated vs. untreated cells) to pinpoint significant AS variations. Accurate transcriptome sequencing is vital for research in drug development and diagnostics. The optimization of sequencing methods is an ongoing endeavour that requires continuous development. A chapter of this thesis compares the impact of omitting polyadenylation (poly(A)) enrichment in Oxford Nanopore Technologies (ONT)’s direct complementary deoxyribonucleic acid (cDNA) sequencing protocol to improve library prep efficiency and sequencing accuracy. Analysis indicated that excluding poly(A) selection does not negatively affect sequencing metrics but enhances read length and enables the sequencing of histone messenger ribonucleic acid (mRNA). Further, significant differences in poly(A) tail lengths between selected and unselected samples were found, suggesting a bias against shorter tails with enrichment. Gene composition and unique gene identification remain consistent across conditions. These findings support the potential advantages of omitting poly(A) enrichment in transcriptome sequencing while advocating for further validation. With optimized methods, the impact of Trastuzumab on AS profiles in BT474 and SKBR3 cell lines was tested. Using ONT long-read sequencing, significant AS events were identified in response to Trastuzumab treatment. The findings reveal differential isoform expression in genes involved in cellular signaling, RNA processing, and stress response pathways. Notably, nuclear paraspeckle assembly transcript 1 (NEAT1) and pre-mRNA processing factor 38B (PRPF38B) exhibited distinct AS patterns across multiple cell lines, suggesting HER2-mediated regulatory mechanisms. This study underscores the utility of ONT sequencing and high throughput data processing as an effective and efficient means for explaining complex AS landscapes affected by therapeutic treatments.
Structural and Functional Characterization of a Modular Immunoglobulin A Protease from Thomasclavelia ramosa
(University of Waterloo, 2024-08-29) Tran, Norman
Immunoglobulin A proteases (IgAPs) are a diverse group of enzymes secreted from a wide range of mucosal bacteria. These enzymes have convergently evolved to cleave immunoglobulin A (IgA), the main antibody found on the mucosa, as a means of modulating the bacterium’s relationship with their host tissues. Due to the various biological functions and biochemical properties of these enzymes, the study of IgAPs has provided multifaceted insight into aspects of mucosal immunity, enzyme structure and function, and the structural basis for substrate specificity. Only two of three known IgAP enzyme families have been investigated using an in-depth structural and functional approach. This thesis thus aimed to carry out these analyses on the IgAP from Thomasclavelia ramosa as a representative member of this last poorly characterized family. X-ray crystallographic, small-angle X-ray scattering, and gel-based kinetic techniques were used to reveal that, unlike the other two IgAP families, the T. ramosa IgAP has a truly modular protein architecture that can be split into and produced as distinct minimized domains that retain function. The crystal and solution-scattering structures of various domain constructs were also used to generate a working model for how the T. ramosa IgAP recognizes and has high specificity for IgA. This thesis provides the first in-depth biochemical account of this IgAP family and pave the way for advances in clinically relevant IgAP-related research and our understanding of IgAPs as a whole.
Wetland hydrology and the impacts of beaver dams in the Upper Columbia River floodplain wetlands
(University of Waterloo, 2024-08-28) Leven, Catriona
The Upper Columbia River floodplain wetlands are the last remaining undammed stretch of floodplain wetlands along the Columbia River and continue to experience a natural flood pulse. This flood pulse interacts with natural levees and beaver dams across the floodplain and habitat heterogeneity results, with individual wetlands within the Columbia Wetlands having different hydrographs. I conducted research in 38 wetlands from 2020 to 2022 and aimed to determine if differing wetland hydrology allowed for wetland groups to be determined, and if those groups could be attributed to gaps in natural levees and beaver dams. Hydrograph attributes can be used to differentiate wetland groups, with three or four groups being identified depending on year. Random Forest models based on measurements of the levees, levee gaps, and beaver dams had an Out-Of-Box Error Estimate of between 36% and 53% across all groups depending on year, indicating correct classification of between 64% and 47%. Combining hydrograph attributes and levee gap and beaver dam metrics, we can describe these groups on a gradient of connectivity to the Columbia River, being Most Connected, with large open levee gaps, Partially Connected, with levees without gaps or with gaps dammed by beaver dams that are smaller, and Least Connected, with levees without gaps or gaps dammed by beaver dams that are bigger. This demonstrates the large impacts of beavers on shaping wetland systems and has implications for the differing impacts of climate change on these different wetland groups.
Characterization of darter (Etheostoma spp.) interspecific energetic responses to climate change induced temperature changes
(University of Waterloo, 2024-08-27) Weber, Allison
Shallow freshwater ecosystems are predicted to experience increases in temperature variability as the occurrence and severity of heat waves continues to rise. Ectothermic organisms like fish are especially vulnerable to these acute temperature increases as their physiological functioning is directly regulated by environmental conditions. Thus, understanding their capabilities of responding to thermal stress is critical to predict how these species will be affected by climate change. Here, we characterized the elevated temperature responses of three closely related darter species: Fantail (Etheostoma flabellare; FTD), Rainbow (Etheostoma caeruleum: RBD), and Johnny darter (Etheostoma nigrum; JD) native to the Grand River of Southern Ontario, via three experiments: Experiment #1: assessment of thermal tolerance limits and energetic enzymatic activity, Experiment #2: determination of thermal preference, and Experiment #3: characterization of metabolic responses to elevated temperatures. Specifically, Experiment #1 determined each darter’s CTmax and quantified activity and gene expression of enzymes involved in glycolysis, the Krebs cycle, and the electron transport chain in brain and heart tissue at 15C baseline and at thermal tolerance limits. Experiment #2 determined differences in thermal preference and mobility between species, and Experiment #3 compared darter aerobic scope (AS) during exposure to four different heat ramp exposures designed to mimic previously recorded heat waves. Significant differences were observed in the thermal tolerance limits of each species. For brain tissue, FTD had higher baseline enzymatic activity compared to JD and RBD, however at CTmax, this difference was lost, as both JD and FTD had similarly high enzyme activity relative to RBD. Intraspecifically, JD demonstrated a superior plastic ability, often having a significantly higher enzyme activity at CTmax compared to its baseline counterparts, while RBD activity declined at CTmax with respect to its baseline levels. Heart tissue exhibited no interspecific differences in activity levels at baseline. At CTmax, however, JD had greater activity than RBD for all heart enzymes, although neither JD or RBD were different from FTD. Similar intraspecific trends as brain were observed, with FTD and JD increasing activity at CTmax, and RBD decreasing. No differences were observed in thermal preference between species, although FTD demonstrated significantly higher mobility than JD. Metabolically, FTD AS was significantly greater than JD and RBD at both 25C and 30C, however no differences were observed at 15 or 20C. These results suggest that FTD may be the best equipped at responding to temperature-induced increased metabolic demands due to their higher baseline enzymatic activity and broader aerobic scope. This FTD advantage, and the interspecific differences observed throughout this study, may a be a result of prior adaptation and acclimatization to each species’ respective microhabitat conditions, as it is expected that FTD reside in warm, high flow, and thermally variable regions, JD in warm, moderately thermally variable, low flow environments, and RBD in cold, fast flow, and thermally stable habitats. Exposure to warm and fluctuating habitats, and high mobility levels, have been shown to broaden metabolic function, potentially explaining the high enzyme activity and aerobic scope seen in FTD, defining their superior CTmax. Collectively, these findings provide insight to predict how climate change will affect local species, and may have conservation applications for determining which species may be most at risk with increased occurrence of extreme heat events.
Enhanced Precision of Aerial Herbicide Application for Invasive Species Management
(University of Waterloo, 2024-08-23) Lew-Kowal, Grace
Invasive common reed (Phragmites australis ssp. australis) has established and dominated in Ontario wetlands for decades. The detrimental effects of P. australis invasions on wetland habitats have demanded intervention through aggressive suppression efforts. However, constraints in available control methods to suppress P. australis have led to persistence. To improve P. australis management in wetlands, we investigated remotely piloted aircraft systems (RPASs) as a precision tool for herbicide application. We applied Habitat® Aqua with a spray-equipped RPAS at selected pilot sites, marking the first-ever application of its kind in Canada. We evaluated the suppression efficacy of RPAS-based herbicide application to P. australis and examined the plant community changes one-year after the initial herbicide application. We found a > 99% reduction in live P. australis stems, along with reductions in species richness (33%), Shannon-Weiner diversity (73%), Simpson’s reciprocal diversity (50%), and Pielou’s evenness (73%) in the year following herbicide application. We performed an in-field application experiment to quantify the herbicide deposition of an RPAS-based application through the vertical profile of a P. australis canopy, comparing medium and coarse droplet sizes. We determined that both droplet sizes achieved similar coverage and vertical penetration of herbicide within the P. australis canopy. Lateral droplet drift occurred at least 8 m away from the application area with medium droplets diminishing at a faster rate than the coarse droplets. We used remotely-sensed images taken before and after the RPAS-based herbicide application to determine the application accuracy and post-treatment effects. We determined that RPAS-based herbicide application to P. australis was on-target 91% of the time. The herbicide drift footprint extended up to 20 m away from the application area, representing a distance that is 96% lower than the label-recommended buffer distance for Habitat® Aqua application with a helicopter.
Seasonal temperature induced heart-collagen remodeling response in the rainbow darter (𝘌𝘵𝘩𝘦𝘰𝘴𝘵𝘰𝘮𝘢 𝘤𝘢𝘦𝘳𝘶𝘭𝘦𝘶𝘮)
(University of Waterloo, 2024-08-22) Hamel, Michael
Acclimation to temperature changes in fish has been shown to prompt a cardiac remodeling response, with collagen protein playing a key role, although the mechanism of this response remains unclear. Currently, it is believed to be a seasonal adaptation to shifting temperatures, with studies indicating that microRNA-29b (miR-29b), an epigenetic non-coding RNA, targets collagen mRNA in the heart. To further explore these questions, this thesis characterizes the remodeling response in a wild population of rainbow darters (Etheostoma caeruleum) to examine seasonal effects in a natural environment, which moves beyond the current lab-only studies. Rainbow darter heart tissue was collected on-site at three season timepoints (Spring, Summer, Fall 2023) from the Grand River, Grand Valley, ON. Water temperature loggers were deployed to monitor the fluctuating river temperatures throughout the study. Gene expression of miR-29b, and the three collagen type I protein transcripts (col1a1, col1a2, & col1a3) was measured in heart tissue through RT-qPCR. Hearts were embedded for histological analysis to visualize heart morphology and quantify the collagen protein content through picro-sirius red staining. Additionally, western blot analysis was performed to measure collagen type I abundance for each season. Temperature loggers revealed substantial daily water temperature fluctuations, with differing fluctuation profiles dependent on season. Results from RT-qPCR revealed seasonal differences in expression of col1a1, and col1a2, and western blot revealed a season effect driving a trend in collagen type I protein differences, suggesting the presence of this remodeling response in a non-model species. Although only a sex-specific difference was found in miR-29b expression and no significant regression was found between miR-29b and any of the collagen type I mRNA, further investigation into its role in this remodeling in vivo will be required. The lack of a compact myocardium layer was determined from cardiac tissue sections, which previously had not been investigated in the rainbow darter and evidence of cardiac hypertrophy at acclimatization to colder temperatures was demonstrated by higher relative heart mass compared to body mass in spring and fall, compared to summer. Understanding the impacts of temperature fluctuations and extreme weather events on local fish populations is increasingly crucial. With limited studies of cardiac collagen remodeling in natural environments, it is important that there be continued focus on bringing elements of natural seasonality into the context of lab studies to further investigate this remodeling response as seasonal plasticity and flexibility. This thesis contributes to a more comprehensive understanding of seasonal effects in a natural environment.
The Effect of Acute Heat Stress on the Chinook Salmon Immune System and on Ability to Combat 𝘝𝘪𝘣𝘳𝘪𝘰 𝘢𝘯𝘨𝘶𝘪𝘭𝘭𝘢𝘳𝘶𝘮 Infection
(University of Waterloo, 2024-08-13) McKenzie, Emily
The frequency, duration, and intensity of heatwaves in western Canada is expected to rise in the coming years. As a result, shallow rivers and streams have also experienced drastic changes, and water temperature may fluctuate by up to 13 °C throughout the duration of a single day. This poses a problem for salmon species, as they travel from marine to freshwater environments to spawn. Additionally, salmon are poikilothermic, meaning their physiological functions are influenced by their surrounding water temperature. Encountering water that is outside of their optimal growth temperature of 11-17 °C may result in behavioural and metabolic changes and, in extreme cases, rapid death. This may also result in increased susceptibility to infectious disease, as warming waters may render them temporarily immunocompromised and increase the virulence factors of some pathogens. Salmon have several cellular mechanisms to survive these stressful events, including heat shock proteins, immune responses, and secretion of glucocorticoids. Interestingly, the concept of thermal preconditioning is emerging as a method to aid salmonids’ ability to handle increasing water temperatures – exposure to a controlled, short term stimulus triggers a physiological response that prepares them for future, more extreme stress exposure. However, thermal preconditioning has never been tested before exposure to a pathogen. Further, the immune effects of heat waves have scarcely been investigated in salmon. In this study, we performed an environmentally applicable mock heat shock on juvenile Chinook salmon (Oncorhynchus tshawytscha) and measured their immunological responses up to 14 days afterwards. Chinook salmon also received an injection of live Vibrio anguillarum after heat shock to determine if heat deterred their ability to fight a systemic bacterial infection. The transcripts of IL-1β, IL-8, TNF-α, IL-10, TGF-β, IFN-γ, IL-2, cathelicidin, hepcidin, MHC1α, tapasin, MHC2α, MHC2β, HSP47, HSP70, and HSP90 were quantified by qPCR in the spleen, gills, and hindgut, as well as assessment of the stress response by measuring glucose, lactate, cortisol, and HSP47 levels in the plasma. Heat shock did not affect mortality rates due to vibriosis compared to salmon that received V. anguillarum alone. Additionally, heat shock mitigated the pro-inflammatory and corresponding anti-inflammatory responses needed to combat infection by initially upregulating il1b, tnfa, il8, and il10, then returning to normal levels by three days post-infection. The antimicrobial peptides cathelicidin and hepcidin played a significant role in combatting infection in both V. anguillarum treatment groups. Transcript levels of hsp47 and hsp90 were upregulated in response to both heat shock and bacterial infection; however, hsp70 expression was surprisingly low through the duration of the trial. HSP47 proteins measured by ELISA in the serum did not differ between any treatment groups. Heat shock caused a significant increase in plasma and cortisol lactate concentrations that both returned to basal levels 6-hours post-heat shock. Altogether, these data indicate that the experimental heat shock had a positive preconditioning effect on Chinook salmon and provides new insights on the interactions between the host, environment, and pathogen over a 14-day period. This research also provides a first step in understand how increasing river temperatures affect the salmon immune and stress responses and can help inform decision making in policy and sustainability initiatives to protect Pacific salmon populations in the coming years.
Early life exposure to diel thermal variation alters microRNA expression and performance in zebrafish.
(University of Waterloo, 2024-08-12) Gavarikar, Sana Manish
Freshwater ecosystems are characterized by large thermal variations, especially in the summer months. However, this variability is rarely accounted for in laboratory studies examining physiological processes of fish inhabiting these environments, thereby producing results which lack ecological realism. Understanding how animals respond to and cope with rapid changes in temperature is more crucial than ever as the range of thermal fluctuations in these habitats is expected to increase dramatically due to increases in the frequency and severity of heat waves. Recently, it was posited that epigenetic mechanisms could buffer fish against such thermal fluctuations as they act on a more rapid timescale than genetic adaptation. Thus, the aim of this thesis was to understand how chronic exposure to thermal variability impacts the physiology of freshwater fish, and if these effects are associated with changes in epigenetic modulators called microRNAs (miRNAs) which act by repressing the translation of target genes. This was achieved by subjecting zebrafish (Danio rerio) to realistic diel thermal fluctuations (FLUX; 28 ± 5°C) throughout the embryonic and larval stages to evaluate the effects on physiological processes like metabolism and survival, and on the expression of seven thermosensitive miRNAs and three heat shock proteins (HSPs). These fish were compared to those kept at constant control (CTRL; 28°C) and constant elevated (HEAT; 33°C) conditions to allow for comparisons with fish reared under optimal lab conditions and in traditional climate change experiments, respectively. After the developmental stages, the fish from CTRL and FLUX treatments were reared under common control conditions until adulthood to understand if developmental exposure to fluctuations altered miRNA expression profiles in the brain and the upper thermal tolerance (CTmax). This study revealed that while the HEAT conditions reduced survival throughout development, the FLUX thermal regime had no impact on this. Additionally, both FLUX and HEAT conditions significantly altered body weight as well as the miRNA expression profiles during early life stages. Although juveniles from both FLUX and HEAT conditions were able to metabolically compensate to their respective thermal regimes, the degree of compensation was greater in FLUX fish. I found that miR-181a-5p, which regulates pathways associated with mitochondrial biogenesis and respiration, was significantly upregulated in the juveniles from these groups, suggesting that changes in this miRNA could modulate the observed metabolic impacts. Notably, this miRNA remained elevated in the brains of longitudinal adults with FLUX developmental histories, though their CTmax was unaffected. Besides its role in modulating mitochondrial function, insights from mammalian models suggest that this miRNA can also enhance neuronal injury/damage in vertebrate brains. This could indicate that FLUX fish have an altered capacity for recovery following acute thermal stress. Collectively, the findings presented in this thesis demonstrate that developmental plasticity may be regulated by changes in epigenetic processes and underscore the need to incorporate variability into our experiments, as it produces a robust and long-lasting impact on the physiology of fish that is distinct from static temperature exposures. Ultimately, ecologically realistic conditions and the plasticity potential of populations need to be accounted for in investigations to accurately predict how fish will respond to the multiple stressors caused by climate change.
Investigation of the atypical type III secretion system in Pseudomonas syringae strains using long read sequencing technology
(University of Waterloo, 2024-08-12) Lorv, Janet
Ubiquitous across many environments, Pseudomonas syringae is a phytopathogen that is spread through the water cycle. With a broad host range, Pseudomonas syringae has been reported to cause outbreaks across many agricultural crops including potato, mango, and kiwifruits. Although this species is highly studied, interest in agricultural impacts has meant that sequencing efforts have focused primarily on virulent strains isolated from diseased plants. In this work, we explored the genetic differences of a recently diverged, closely related and putatively non-virulent (i.e., not visibly detected by host plant) subphylogroup of P. syringae strains, subphylogroup 2c. Strains in this monophyletic group are a part of the larger virulent phylogroup 2 and more broadly grouped with other agriculturally virulent strains in phylogroups 1 and 3. Virulence of this bacterial species complex has been attributed to the genetic mobility of genes encoding type III effector proteins that are secreted by the type III secretion system. Gain or loss of these genes can cause P. syringae strains to switch between virulent and avirulent (i.e., detected by host plant and rendered not virulent) phenotypes. Many other niche adaption genes such as phytotoxin biosynthesis genes can also contribute to a virulence phenotype. We propose that the mechanisms contributing to the putative non-virulence phenotype of subphylogroup 2c may be elucidated via identification and examination of the divergent gene clusters within P. syringae strains. To capture the full complement of genes, we sequenced and assembled complete genomes of two subphylogroup 2c strains, a biocontrol strain P. syringae pv. syringae 508, and a strain isolated from a healthy leaf, P. syringae TLP2. These strains were sequenced using Oxford Nanopore Technology's MinION long read sequencer to generate complete genomes without additional sequencing. Using one strain per R9.4 flowcell, we were able to generate >300x coverage for each strain with maximum read lengths exceeding 500kb and 250kb respectively allowing easy assembly of the full genome lengths. However, the ease-of-use of nanopore sequencing is hampered somewhat by the relatively high sequencing error rate of the reads, requiring extensive testing and optimization of recently developed software to maximize assembly quality. Despite optimization, many genes in the initial assembly remained fragmented. To address this issue, we developed the software tool Kastor that identifies and corrects draft assembly errors through comparisons to reference genomes and/or protein sequence information. Users can reference related genomes for more precise error identification, or use reference proteins for a more generalized identification method that doesn't rely on data from closely related species. For both the Pss508 and PsyTLP2 sequenced genomes, fewer than 3000 errors (comprising <0.05% of the assemblies) were corrected, however assembly completeness was increased from <85% to >99.8% as benchmarked using single copy orthologs. The completed assemblies were then annotated using NCBI's PGAP. The annotated gene sets of the two P. syringae strains were then compared to 428 P. syringae strains across the species complex. We identified several divergent gene clusters in both sequenced and subphylogroup 2c genomes, with the most prominent diverging gene cluster set encoding the atypical variant of the type III secretion system (T3SS). Using PIRATE, we identified a related gene cluster in the distantly related phylogroup 13. With the analysis of both gene sequence and gene content, we propose that the atypical variant is a result of horizontal transfer and not divergence from the canonical variant in closely related strains. The horizontal acquisition of this T3SS variant in the most recent common ancestor of subgroup 2c likely triggered the divergence of the subgroup from group 2, the loss of common type III effector genes, and a likely niche/virulence change, resulting in putatively weakly-virulent strains.
Wastewater Surveillance of Influenza (A, B) and Respiratory Syncytial Virus (RSV) in Southern Ontario
(University of Waterloo, 2024-07-16) Abu Farah, Joud
Wastewater-based surveillance (WBS) of SARS-CoV-2 has been highly effective at tracking trends of COVID-19 infections across the globe. This success led to the question of the possible applicability of WBS in monitoring other respiratory viruses that are also posing a concern for healthcare systems. Traditional influenza and RSV surveillance consists of monitoring hospital admissions, clinical testing data and outpatient visits. Even when clinical surveillance and hospitalization admissions are used to monitor the spread of these infections, they are often underestimated, with a lag between detection and community spread. In this study, wastewater surveillance of influenza A, influenza B and respiratory syncytial virus (RSV) was investigated, by quantifying the viral RNA of these viruses in wastewater and comparing these trends to clinical metrices. The solid–liquid partitioning behaviors of influenza A, influenza B, RSV and SARS-CoV-2 in wastewater were examined to understand the behavior of the viruses in wastewater. Analyzing whether each virus is present in the liquid or solid fraction of wastewater may affect the data interpretation and inform further method development. The viral RNA in the liquid and solid fractions were separated and enriched by undergoing different centrifugation settings, overnight polyethylene glycol (PEG) precipitation followed by centrifugation, or ultrafiltration using a Centricon Plus-70 device. The influenza A, influenza B and RSV viral RNA concentrations were found to almost exclusively partition in the solids fraction of wastewater, which increased with an increase in centrifugal settings, unlike SARS-CoV-2 that had a more even split in signal between the solids and liquid fractions. The effectiveness of normalization using the endogenous pepper mild mottled virus (PMMoV) was also examined, by comparing the normalized influenza signal with the raw signal. Normalization using the PMMoV biomarker did not increase or hinder the correlation with clinical testing data relative to the raw influenza signal in wastewater. Wastewater samples were collected once weekly from two wastewater treatment plants in the Region of Waterloo, Canada, from September 15, 2022, to June 21, 2023. Wastewater was treated overnight with polyethylene glycol (PEG), centrifuged (12,000g 1.5 h), then extracted (pellet) with Qiagen RNeasy PowerMicrobiome Kits and quantified using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). The RNA concentrations of influenza A, influenza B and RSV in wastewater strongly correlated with the cases reported by episode date and hospitalizations, in the cities of Kitchener and Waterloo (Spearman’s Rank correlation coefficient rho ranging from 0.41-0.85). Wastewater surveillance can be a very effective additional surveillance tool to support public health officials in monitoring the trends of respiratory viruses in communities. Further development of WBS for respiratory virus monitoring will help public health to better prepare for these and other emerging pathogens in the future.
Degradation of Polyethylene Terephthalate (PET) and Polyamide (PA)
(University of Waterloo, 2024-07-16) Griffiths, Erin
Microplastics have become an increasing concern to humans and ecosystems as plastic production continues to soar, due to their prevalence in the environment and lifespan. Plastic is cheap and durable making it an ideal industrial and commercial material. However, because of this popularity, it resides in most places on earth, including in human blood, and is difficult to remove due to its small size. These plastics can enter the environment through numerous methods, from landfills and dumps to washing machines and sinks. In recent years, there has been significant investigation in reducing plastic pollution. This a difficult task attributed to the varying size, shape, polymer type, chemical properties and location plastic can be found. It’s critical to understand the rate of degradation and the factors that influence it for two main reasons; it provides accurate timelines of degradation and techniques that may increase degradation need a starting point. In Chapter 2, I investigate the degradation rate of laboratory grade polyethylene terephthalate (PET) using a model enzyme (Huimcola insolens cutinase) to hydrolyze the plastic. This research aims to characterize the polymers used such that results can be compared and identify the analyses which capture degradation and characterize the polymer best. Environmental factors controlling enzymatic plastic degradation are not well studied and this experiment aimed to study the effect of incubation temperature, exposure to freeze-thaw cycles (FTCs) and extreme temperatures on the degradability of laboratory-grade PET. In addition, we also assessed the degradability of consumer-grade PET, sourced from plastic bottles, for comparison to the laboratory-grade PET. The first test was under variable temperatures, where plastic was incubated at 25 ˚C, 40 ˚C and 55 ˚C. The results show increased temperatures increase the rate of polymer degradation. The second set of tests were conducted under different pretreatments; treatments the plastic would undergo before incubation at 40 ˚C. Plastic was exposed to a series of freeze-thaw cycles (FTCs) or extreme temperatures (-70 ˚C or +55 ˚C). It was found any type of pretreatment increased the rate of degradation compared to plastics that did not undergo any pretreatment. The final condition tested was plastic type, where PET water bottles were obtained and incubated at 55 ˚C to determine the differences in degradability between laboratory-grade PET and consumer-grade PET. Consumer-grade PET was found to not have any significant degradation after 10 weeks of enzyme exposure, raising serious concerns regarding its degradability and lifespan. This result suggests that modifications to the consumer-grade PET during the fabrication process, such as heat treatments, are altering its chemistry and its degradation kinetics. Analyses for degradation and characterizing the polymers included: Fourier-transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and tensile strength measurements. Analysis of the crystallinity, tensile strength, SEM images and FTIR spectra measured indicate that PET’s physical and chemical properties were modified when degraded. Overall, the PET’s tensile strength decreased and the crystallinity increased with increasing hydrolytic degradation. FTIR spectral changes were seen early on, with peaks of interest at 1237 cm-1, 1016 cm-1 and 1087 cm-1, and finally at 1716 cm-1, and the flattening of these peaks increased with increasing hydrolytic degradation. The results highlight that enzymatic degradation rates can be highly variable due to differences in environmental conditions. It also highlights the large difference in the degradability of consumer-grade versus laboratory-grade PET, which has significant implications for in situ environmental degradation rates. In Chapter 3, I investigated the rate of laboratory-grade PET and polyamide (PA) degradation in stormwater pond sediment over a 16 month period in a stormwater pond in Kitchener, Ontario. Microplastic accumulation in the environment, especially in bodies of water and sediment is a well-known problem. Stormwater ponds act as a microplastic sink and draw pollutants from urban and industrial wastewater before it enters oceans or lakes. This results in high levels of microplastics remaining in stormwater pond sediment. Stormwater ponds are an excellent site to determine realistic plastic degradation in the environment, in a contained area where high concentrations of plastic is known to be present. To date, no long-term polymer degradation studies have been conducted in a stormwater pond despite the rising popularity of these ponds. For this study, 8 pore water samplers (peepers) were packed with pond sediment and plastic pieces were inserted into each cell of the peeper. An additional 8 peepers filled with water, such that pore water chemistry could be collected. The peepers were inserted into the pond sediment and sacrificed periodically over the course of 16 months. For the first 8 months both PET and PA plastic increased in mass as they absorbed water. After 16 months of field incubation, PA had degraded by 0.42% and PET was still net positive (higher mass than before the incubation) however it was close to its original weight. The obtained results highlight the lack of degradation to plastics in stormwater pond sediment and suggest lifespans are longer than previously estimated. Based on previous degradation studies under sediment conditions, this study suggests that stormwater pond sediment is the least effective at degradation polymers, which may be attributed to the pond water chemistry and microbial communities present. Microplastics are known to accumulate in stormwater pond sediment but they are found to degrade at slower rates than other sediment profiles. The laboratory experiment results in Chapter 2 show under ideal conditions laboratory-grade PET degrades minimally at low temperatures. Additionally, the lack of degradation seen with the consumer-grade PET in Chapter 2 suggests that under environmental conditions, the polymer would take even longer than the laboratory-grade polymers to degrade. The combination of Chapter 2 and 3 demonstrate the difference between ideal and environmental conditions for polymer degradation. This research provides evidence to strongly advocate for the removal of microplastics before they enter the environment as I have proven they take considerable lengths of time to degrade under various conditions. I encourage this research to be used by any future researchers who hope to develop methods for plastic pollution reductions.

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